Efficient preparation method of intermediate of heterocyclic drugs

Abstract

The invention discloses an efficient preparation method of an intermediate of heterocyclic drugs. According to the efficient preparation method of the intermediate of the heterocyclic drugs, the intermediate of the heterocyclic drugs are produced by catalyzing a heterocyclic substrate by coupling an alcohol dehydrogenase mutant and glucose dehydrogenase; and the alcohol dehydrogenase mutant is prepared by mutating tyrosine at position 127 of a alcohol dehydrogenase female-parent with an amino acid sequence shown as SEQ ID No. 2 into tryptophan. The alcohol dehydrogenase mutant, namely mutant Y127W, utilized in the efficient preparation method of the intermediate of the heterocyclic drugs is capable of reducing product inhibition effects in a single aqueous system without addition of any co-solvent, thereby achieving a conversion rate higher than 99% within 12 hours. Being coupled with the glucose dehydrogenase (BmGDH), the mutant Y127W is capable of realizing gram-level preparation ata scale of 50 ml in a single aqueous system without addition of any exogenous coenzyme or organic co-solvent when substrate concentration is up to 600g x L<-1>; and the catalyst loading capacity is 3.3%. As for a final product, namely (S)-NBHP, the e.e. value is up to 99.4%; the space-time yield is about 1400g x L<-1> x d<-1>; and the product purity is 99.58%.

Description

technical field [0001] The invention relates to a high-efficiency preparation method of a heterocyclic drug intermediate, belonging to the technical field of biocatalysis. Background technique [0002] (S)-N-B OC -3-Hydroxypiperidine [(S)-NBHP] is a key chiral intermediate in the synthesis of the drug Ibrutinib (IBRUTINIB) for the treatment of lymphoma. The method of biosynthesis of (S)-NBHP is more green and environment-friendly, thus attracting more and more attention. However, previous studies have shown that the biosynthesis of (S)-NBHP requires the addition of organic co-solvents and expensive coenzymes, and the high concentration of substrates will lead to substrate or product inhibition, which will increase the cost of (S)-NBHP synthesis. [0003] (1) 2009A CHERETZ et al., used the method of biocatalytic synthesis for the first time, using the reductase in carrot cubes for catalysis. The catalyst was cheap and environmentally friendly, and provided a new idea for t...

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Problems solved by technology

[0013] (11) Patent CN201310173088.2 discloses a method of using recombinant ketoreductase (KRED) enzyme powder to asymmetrically reduce N-B OC -3-piperidone, but did not disclose the gene sequence or amino acid sequence of ketone reductase (KRED)

Patent CN201310054684.9 discloses an asymmetric synthesis of (S)-1-tert-butoxycarbonyl-3-hydroxypiperidine using alcohol dehydrogenase PAR, but the organic reagent isopropanol is used for the coenzyme cycle, and the organic reagent There is a greater degree of damage to the enzyme activity, and it has obvious inhibitory effect

Patent CN201610132936.9 discloses a method using carbonyl reductase R E The CR enzyme asymmetrically reduces ketoreductase (KRED), but this enzyme requires N I -NTA purification, and it uses 2-octanol-water two-phase reaction, which is not conducive to large-scale production or relatively high production cost

Pichia pastoris P reported in patent CN108220358A ICHIA SP.SIT2014 can be used as a biocatalyst for preparing (S)-NBHP, but too much catalyst addition increases the production cost

CN10822061A reports that ketoreductase MT-KRED is used for preparing (S)-NBHP, but needs to add expensive coenzyme in reaction process

[0014] Although the ketoreductase reported above can be used to prepare (S)-NBHP, the reaction process requires the addition of expensive coenzyme, a large amount of enzyme and organic solvents, which is not conducive to the scale-up application in the actual industry.

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